Project Summary/Abstract The proteasome is a multisubunit macromolecular machine that mediates most regulatory protein degradation and removes toxic proteins from cells. It is essential for activities as diverse as the cell cycle, adaptive immunity, and DNA repair. Alterations to proteasome activity impact numerous human diseases, including cancer, neurodegenerative disorders, and diabetes. The proteasome consists of three functional subcomplexes: the lid, the base, and the core particle. Each subcomplex performs distinct functions during substrate degradation. The lid removes the proteasomal targeting signal, the base uses energy from ATP hydrolysis to unfold the substrate, and the core particle then cleaves it into short peptides. These activities?as well as the subcomplexes that harbor them?are intimately linked by static and dynamic inter-subcomplex interactions. Recent structural studies have unexpectedly revealed that the proteasome exists in at least two well-defined conformational states?an apo state, in which the substrate passageways and the enzymatic active sites within these subcomplexes are blocked, and an engaged state, in which these passageways and active sites are opened and aligned, ready to accept and process substrates. Thus, these states reflect ?off? and ?on? conformations for the proteasome, respectively. Proteolytic inhibitors of the proteasome such as Velcade (bortezomib) are proven anticancer drugs, but resistance to these agents is already emerging. This necessitates alternative approaches to control proteasome function. Manipulation of the conformational state of the proteasome could allow for their selective activation or inactivation at will. This strategy could permit treatment of proteasome-addicted cancers via proteasome inactivation, as well as treatment of proteinopathies such as Alzheimer?s and type II diabetes, via enhancement of proteolysis to clear toxic inclusions. The long-term goal of this project is to understand the molecular mechanisms regulating engagement and communication between proteasomal subcomplexes, and how they relate to the proteasome?s conformational state. We seek to determine how individual conformation- specific contacts between lid and base subunits control proteasome structure and function (Aim1), dissect the critical role of nucleotide binding in reorganization of the lid-base interface to promote the engaged state (Aim 2), and to develop small molecules that disrupt lid-base coordination to be used as tools for studying proteasome function in human cells or in vitro (Aim 3). We anticipate our studies will yield insights into allosteric communication, energy use, and substrate processing by the proteasome, as well as yielding new information on proteasome biogenesis and structure. Further, the functional elements of the proteasome are found in many other multiprotein machines, so our studies could reveal general principles governing the function of diverse macromolecular complexes, and thus will impact numerous areas of cell and molecular biology.